Wave Propagation in Lipid Monolayers

Sound waves are excited on lipid monolayers using a set of planar electrodes aligned in parallel with the excitable medium. By measuring the frequency-dependent change in the lateral pressure, we are able to extract the sound velocity for the entire monolayer phase diagram. We demonstrate that this velocity can also be directly derived from the lipid monolayer compressibility, and consequently displays a minimum in the phase transition regime. This minimum decreases from v₀ = 170 m/s for one-component lipid monolayers down to v_m = 50 m/s for lipid mixtures. No significant attenuation can be detected confirming an adiabatic phenomenon. Finally, our data propose a relative lateral density oscillation of Δρ/ρ ∼2%, implying a change in all area-dependent physical properties. Order-of-magnitude estimates from static couplings therefore predict propagating changes in surface potential of 1-50 mV, 1 unit in pH (electrochemical potential), and 0.01 K in temperature, and fall within the same order of magnitude as physical changes measured during nerve pulse propagation. These results therefore strongly support the idea of propagating adiabatic sound waves along nerves as first thoroughly described by Kaufmann in 1989 and recently by Heimburg and Jackson, but already claimed by Wilke in 1912.     

Interactions of Bax and tBid with Lipid Monolayers

The release of cytochrome c from mitochondria to the cytosol is a crucial step of apoptosis that involves interactions of Bax and tBid proteins with the mitochondrial membrane. We investigated Bax and tBid interactions with (i) phosphatidylcholine (PC) monolayer as the main component of the outer leaflet of the outer membrane, (ii) with phosphatidylethanolamine (PE) and phosphatidylserine (PS) that are present in the inner leaflet and (iii) with a mixed PC/PE/Cardiolipin (CL) monolayer of the contact sites between the outer and inner membranes. These interactions were studied by measuring the increase of the lipidic monolayer surface pressure induced by the proteins. Our measurements suggest that tBid interacts strongly with the POPC/DOPE/CL, whereas Bax interaction with this monolayer is about 12 times weaker. Both tBid and Bax interact moderately half as strongly with negatively charged DOPS and non-lamellar DOPE monolayers. TBid also slightly interacts with DOPC. Our results suggest that tBid but not Bax interacts with the PC-containing outer membrane. Subsequent insertion of these proteins may occur at the PC/PE/CL sites of contact between the outer and inner membranes. It was also shown that Bax and tBid being mixed in solution inhibit their insertion into POPC/DOPE/CL monolayer. The known 3-D structures of Bax and Bid allowed us to propose a structural interpretation of these experimental results.     

FCS Analysis of Protein Mobility on Lipid Monolayers

In vitro membrane model systems are used to dissect complex biological phenomena under controlled unadulterated conditions. In this context, lipid monolayers are a powerful tool to particularly study the influence of lipid packing on the behavior of membrane proteins. Here, monolayers deposited in miniaturized fixed area-chambers, which require only minute amounts of protein, were used and shown to faithfully reproduce the characteristics of Langmuir monolayers. This assay is ideally suited to be combined with single-molecule sensitive fluorescence correlation spectroscopy (FCS) to characterize diffusion dynamics. Our results confirm the influence of lipid packing on lipid mobility and validate the use of FCS as an alternative to conventional surface pressure measurements for characterizing the monolayer. Furthermore, we demonstrate the effect of lipid density on the diffusional behavior of membrane-bound components. We exploit the sensitivity of FCS to characterize protein interactions with the lipid monolayer in a regime in which the monolayer physical properties are not altered. To demonstrate the potential of our approach, we analyzed the diffusion behavior of objects of different nature, ranging from a small peptide to a large DNA-based nanostructure. Moreover, in this work we quantify the surface viscosity of lipid monolayers. We present a detailed strategy for the conduction of point FCS experiments on lipid monolayers, which is the first step toward extensive studies of protein-monolayer interactions.     

Accessibility of Calcium Ions to Anionic Sites of Lipid Monolayers

ALTHOUGH the calcium ion is believed to play a functional role in various membrane phenomena such as nervous excitation, muscular contraction and cell permeation by interacting with the reactive sites of the membrane, little is known about the binding characteristics of calcium with the anionic sites of the well specified lipid membrane^1,2.     

Pentameric Two-Dimensional Crystallization of Rabbit C-Reactive Protein on Lipid Monolayers

As a member of the pentraxin family, C-reactive protein plays various roles in the nonspecific immunity of animals. Though soluble, C-reactive protein always functions on membranes. In order to study the structure of the membrane-bound protein and the reaction between protein and membranes, two-dimensional (2D) crystallization of rabbit C-reactive protein on lipid monolayers was performed. The 2D crystals composed of pentameric proteins were obtained on lipid monolayers by specific adsorption for the first time. The projection map at 26-A resolution is presented, which exhibits P2 symmetry with lattice parameters a = 158(+/-3) A, b = 92(+/-1) A, and gamma = 107(+/-1) degrees. The current work may give a basis for the further study on the structure of complexes made up of C-reactive protein with its functional binding molecules on membranes.     

Diffusion and Partitioning of Fluorescent Lipid Probes in Phospholipid Monolayers

The pressure-dependent diffusion and partitioning of single lipid fluorophores in DMPC and DPPC monolayers were investigated with the use of a custom-made monolayer trough mounted on a combined fluorescence correlation spectroscopy (FCS) and wide-field microscopy setup. It is shown that lipid diffusion, which is essential for the function of biological membranes, is heavily influenced by the lateral pressure and phase of the lipid structure. Both of these may change dynamically during, e.g., protein adsorption and desorption processes. Using FCS, we measured lipid diffusion coefficients over a wide range of lateral pressures in DMPC monolayers and fitted them to a free-area model as well as the direct experimental observable mean molecular area. FCS measurements on DPPC monolayers were also performed below the onset of the phase transition (Π < 5 mN/m). At higher pressures, FCS was not applicable for measuring diffusion coefficients in DPPC monolayers. Single-molecule fluorescence microscopy and differential scanning calorimetry clearly showed that this was due to heterogeneous partitioning of the lipid fluorophores in condensed phases. The results were compared with dye partitioning in giant lipid vesicles. These findings are significant in relation to the application of lipid fluorophores to study diffusion in both model systems and biological systems.     

The SU Glycoprotein 120 from HIV-1 Penetrates into Lipid Monolayers Mimicking Plasma Membranes

Increasing evidence suggests that the HIV envelope binds through its surface (SU) gp120 not only to receptors and coreceptors, but also to other components of the cellular membrane where the glycolipids appear to be good candidates. To assess the ability of HIV-1 SU gp120 to penetrate into phospholipid membranes, we carried out a study of the interactions between a recombinant SU gp120 from HIV-1/HXB2 and artificial lipid monolayers mimicking the composition of the outer leaflet of the lymphocytes and which were spread at the air-water interface. We show that the protein, in its aggregated form, has amphipathic properties and that the insertion of this amphipathic species into lipids is favored by the presence of sphingomyelin. Furthermore, cholesterol enhances the penetration into mixed phosphatidylcholine-sphingomyelin monolayers. Coexistence of different physical states of the lipids and thus of domains appears to play a major role for protein penetration independently of the presence of receptors and coreceptors. Received: 24 April 2000/Revised: 11 July 2000     

The Mechanism of Detergent Solubilization of Lipid Bilayers

Multiple data are available on the self-assembly of mixtures of bilayer-forming amphiphiles, particularly phospholipids and micelle-forming amphiphiles, commonly denoted detergents. The structure of such mixed assemblies has been thoroughly investigated, described in phase diagrams, and theoretically rationalized in terms of the balance between the large spontaneous curvature of the curvophilic detergent and the curvophobic phospholipids. In this critical review, we discuss the mechanism of this process and try to explain the actual mechanism involved in solubilization. Interestingly, membrane solubilization by some detergents is relatively slow and the common attribute of these detergents is that their trans-bilayer movement, commonly denoted flip-flop, is very slow. Only detergents that can flip into the inner monolayer cause relatively rapid solubilization of detergent-saturated bilayers. This occurs via the following sequence of events: 1), relatively rapid penetration of detergent monomers into the outer monolayer; 2), trans-membrane equilibration of detergent monomers between the two monolayers; 3), saturation of the bilayer by detergents and consequent permeabilization of the membrane; and 4), transition of the whole bilayer to thread-like mixed micelles. When the detergent cannot flip to the inner monolayer, the outer monolayer becomes unstable due to mass imbalance between the monolayers and inclusion of the curvophilic detergent molecules in a flat surface. Consequently, the outer monolayer forms mixed micellar structures within the outer monolayer. Shedding of these micelles into the aqueous solution results in partial solubilization. The consequent leakage of detergent into the liposome results in trans-membrane equilibration of detergent and subsequent micellization through the rapid bilayer-saturation mechanism.     

The Mechanism of Iron (III) Stimulation of Lipid Peroxidation

A study conducted on Fe²⁺ autoxidation showed that its rate was extremely slow at acidic pH values and increased by increasing the pH; it was stimulated by Fe³⁺ addition but the stimulation did not present a maximum at a Fe²⁺/Fe³⁺ ratio approaching 1:1. The species generated during Fe³⁺-catalyzed Fe²⁺ autoxidation was able to oxidize deoxyribose; the increased Fe²⁺ oxidation observed at higher pHs was paralleled by increased deoxyribose degradation. The species generated during Fe³⁺-catalyzed Fe²⁺ autoxidation could not initiate lipid peroxidation in phosphatidylcholine liposomes from which lipid hydroperoxides (LOOH) had been removed by treatment with triph-enylphosphine. Neither Fe²⁺ oxidation nor changes in the oxidation index of the liposomes due to lipid peroxidation were observed at pHs where the Fe³⁺ effect on Fe²⁺ autoxidation and on deoxyribose degradation was evident. In our experimental system, a Fe²⁺/Fe³⁺ ratio ranging from 1:3 to 2:1 was unable to initiate lipid peroxidation in LOOH-free phosphatidylcholine liposomes. By contrast Fe³⁺ stimulated the peroxidation of liposomes where increasing amounts of cumene hydroperoxide were incorporated. These results argue against the participation of Fe³⁺ in the initiation of LOOH-independent lipid peroxidation and suggest its possible involvement in LOOH-dependent lipid peroxidation.     

Differential Dichrome Staining of Tissue Culture Monolayers: Alternate Dyes and Possible Mechanism

A polyacid-dependent dichrome has been devised which will differentiate epithelial from mesenchymal cells in young dividing primary cultures. Epithelial cells and colonies and nuclei are stained with metanil yellow, the stain is fixed and differentiated with phosphotungstic acid, and the mesenchymal elements are stained with toluidine blue. Several other dyes are tested for substitution in this method. Biebrich scarlet and aniline blue could be substituted for the metanil yellow; Bismarck brown T, Janus green B, crystal violet, and neutral red could be substituted for the basic dye.     

Carbohydrate Conformation and Lipid Condensation in Monolayers Containing Glycosphingolipid Gb3: Influence of Acyl Chain Structure

Globotriaosylceramide (Gb3), a glycosphingolipid found in the plasma membrane of animal cells, is the endocytic receptor of the bacterial Shiga toxin. Using x-ray reflectivity (XR) and grazing incidence x-ray diffraction (GIXD), lipid monolayers containing Gb3 were investigated at the air-water interface. XR probed Gb3 carbohydrate conformation normal to the interface, whereas GIXD precisely characterized Gb3’s influence on acyl chain in-plane packing and area per molecule (APM). Two phospholipids, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), were used to study Gb3 packing in different lipid environments. Furthermore, the impact on monolayer structure of a naturally extracted Gb3 mixture was compared to synthetic Gb3 species with uniquely defined acyl chain structures. XR results showed that lipid environment and Gb3 acyl chain structure impact carbohydrate conformation with greater solvent accessibility observed for smaller phospholipid headgroups and long Gb3 acyl chains. In general, GIXD showed that Gb3 condensed phospholipid packing resulting in smaller APM than predicted by ideal mixing. Gb3’s capacity to condense APM was larger for DSPC monolayers and exhibited different dependencies on acyl chain structure depending on the lipid environment. The interplay between Gb3-induced changes in lipid packing and the lipid environment’s impact on carbohydrate conformation has broad implications for glycosphingolipid macromolecule recognition and ligand binding.     

Carbohydrate Conformation and Lipid Condensation in Monolayers Containing Glycosphingolipid Gb3: Influence of Acyl Chain Structure

Globotriaosylceramide (Gb3), a glycosphingolipid found in the plasma membrane of animal cells, is the endocytic receptor of the bacterial Shiga toxin. Using x-ray reflectivity (XR) and grazing incidence x-ray diffraction (GIXD), lipid monolayers containing Gb3 were investigated at the air-water interface. XR probed Gb3 carbohydrate conformation normal to the interface, whereas GIXD precisely characterized Gb3’s influence on acyl chain in-plane packing and area per molecule (APM). Two phospholipids, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), were used to study Gb3 packing in different lipid environments. Furthermore, the impact on monolayer structure of a naturally extracted Gb3 mixture was compared to synthetic Gb3 species with uniquely defined acyl chain structures. XR results showed that lipid environment and Gb3 acyl chain structure impact carbohydrate conformation with greater solvent accessibility observed for smaller phospholipid headgroups and long Gb3 acyl chains. In general, GIXD showed that Gb3 condensed phospholipid packing resulting in smaller APM than predicted by ideal mixing. Gb3’s capacity to condense APM was larger for DSPC monolayers and exhibited different dependencies on acyl chain structure depending on the lipid environment. The interplay between Gb3-induced changes in lipid packing and the lipid environment’s impact on carbohydrate conformation has broad implications for glycosphingolipid macromolecule recognition and ligand binding.     

The Basic Principles of Collapse Therapy

Collapse therapy has a definite objective of resting and healing tuberculous lesions. It should be used, not so much as an independent means, but as a supplement to other established practices in the treatment of tuberculosis.The indications for each measure have changed over the years and are still changing, so that proper treatment can be given only by those having a knowledge of the changing trends, as well as of the disease and principles of treatment. Each case should be handled on its merits and the choice of procedure should depend generally upon the circumstances, such as the skill of the physician carrying it out, the facilities for the care of the patient, and the equipment for working. Above all, it should be recognized that knowledge, experience, and especially wisdom will go far towards achieving good results.     

微藻油脂合成代谢途径及调控机制的研究进展

随着经济的快速发展,各国对石油的需求仍有增无减,但是石油作为不可再生能源不利于可持续发展。相比之下,生物柴油应运而生且已发展到了第三代,而微藻作为第三代生物柴油的主角因为具有生长速度快,不占用耕地等优势,已逐渐成为具有极大发展潜力的能源原料,所以利用微藻生产生物柴油的技术近年来也成为研究的重点。本综述针对微藻容易进行基因改造的优势,综述了国内外对微藻油脂代谢通路的研究现状及进展,讨论了目前的基因改造方法对通路中关键酶产生的影响,以求找到能有效并稳定增强微藻油脂代谢途径的方法,为以后的研究提供理论指导。     

The Role of Lipid Physical Properties in Lipid Barriers

SYNOPSIS.The hydrophobic nature of lipids means that they providegood bar-riers to the movement of charged and polar molecules.Barrier function appears to depend on the physical state ofthe lipids. Two well-investigated examples in-cludecell membranesand epicuticular lipids of arthropods. Ecologically relevantchanges in temperature significantly affect lipid properties,and both evolutionary and acclimatory differences in lipid compositionappear to preserve the physical properties of lipids under differentenvironmental conditions. These differences are generally believedto be beneficial to the organism, but rigorous examination oftheir adaptive significance is rare. Important issues are howlipid properties are regulated; which properties are physiologicallyrelevant, how are these properties sensed, and what biochemicaland molecular mechanisms regulate lipid properties? Progresshas recently been made in understanding how membrane lipid propertiesare regulated, but regulatory mechanisms for cuticular lipidsand other lipid sys-tems remain unknown.     

The Lipid World

The continuity of abiotically formed bilayer membraneswith similar structures in contemporary cellular life,and the requirement for microenvironments in whichlarge and small molecules could be compartmentalized, support the idea that amphiphilic boundary structurescontributed to the emergence of life. As an extensionof this notion, we propose here a `Lipid World'scenario as an early evolutionary step in theemergence of cellular life on Earth. This conceptcombines the potential chemical activities of lipidsand other amphiphiles, with their capacity to undergospontaneous self-organization into supramolecularstructures such as micelles and bilayers. Inparticular, the documented chemical rate enhancementswithin lipid assemblies suggest that energy-dependentsynthetic reactions could lead to the growth andincreased abundance of certain amphiphilic assemblies.We further propose that selective processes might acton such assemblies, as suggested by our computersimulations of mutual catalysis among amphiphiles. Asdemonstrated also by other researchers, such mutualcatalysis within random molecular assemblies couldhave led to a primordial homeostatic system displayingrudimentary life-like properties. Taken together,these concepts provide a theoretical framework, andsuggest experimental tests for a Lipid World model forthe origin of life.     

Mechanism and Determinants of Amphipathic Helix-Containing Protein Targeting to Lipid Droplets

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Monolayers of ether lipids from archaebacteria

The surface behavior of six different ether lipids from archaebacteria, based on condensation of glycerol or more complex polyols with two isoprenoid alcohols at 20 or 40 carbon atoms, was investigated in monolayers at the air-water interface.The compounds with no complex polar group (GD, GDGT, GDNT) form monolayers showing a reversible collapse at surface pressure as low as 22 dynes/cm. This collapse pressure decrease with temperature in such a way that the film tension remains constant. In condensed films, these molecules do not assume a completely upright position.Lipids with complex polar ends (HL, GLB, PLII) form films more stable to compression. Forcearea characteristics and surface moment values of HL monolayers are similar to those of analogous ester lipids with fatty acid chains. Monolayers of the two bipolar lipids, GLB and PLII, at room temperature present a more condensed state, probably due to the lateral cohesion between long alkyl chains, but a lower collapse pressure.For all bipolar lipids, the area expansion induced by temperature increase is larger than that of monopolar ones.Abbreviations GD Glycerol diether (2,3-di-O-phytanyl-sn-glycerol - GDGT Glycerol-dialkyl-glycerol tetraether - GDNT Glycerol-dialkyl-nonitol tetraether - GLB Glycolipid B - PLII Phospholipid II - HL Total lipid extract from Halobacterium halobium